Inclusion complexes containing an active substance, a cyclodextrin and an agent for interaction

ABSTRACT

The invention relates to a method for preparing a soluble inclusion complex comprising one or several active substances which are hardly soluble in an aqueous medium and are included in one or several host molecules, characterised in that it comprises the following succesive stages: (a) one or several active substances are brought into contact with one or several host molecules; (b) initiation of a molecular diffusion stage by bringing a dense pressurized fluid into contact with the mixture obtained in stage (a) in a static mode in the presence of one or several diffusing agents; (c) recovery of the active substance- host molecule molecular complex thus formed; (d) initiation of a stage wherein an interaction agent is added to and mixed with the active substance host molecule molecular complex; (e) recovery of the soluble inclusion compound thus formed. The invention also relates to the soluble inclusion compound which can be obtained by said method, particularly a piroxicam-cyclodextrin-arginine compound.

The present invention relates to a process for the preparation ofsoluble inclusion compounds by the dense pressurized fluids technology,in particular that of CO₂.

Numerous active substances, in particular of interest in thepharmaceutical field, exhibit a very low solubility or are insoluble inwater and consequently in biological liquids. This implies a lowbioavailability of these active substances and a large increase in thedoses administered to the patients in order to achieve the therapeuticobjective set and thus an increase in the possible side effects relatedto the medical treatments.

The pharmacokinetic properties of a given substance depend, inter alia,on the affinity of its contact surface for the solvent underconsideration, namely water in the case of the pharmaceutical field. Theincrease in the specific surface of powders makes it possible to improvetheir rate of dissolution. In point of fact, the bioavailability ofactive principles can be greatly increased if their rate of dissolutionis improved. Consequently, the formation of molecular complexes composedof one or more active substances and of one or more host moleculescarefully chosen for its (their) high solubility in biological liquidscan thus make it possible to increase dissolution of the activesubstance or substances in biological liquids.

In the pharmaceutical field, a number of patents and publications existrelating to the formation of cyclodextrin-based complexes in thepresence of an agent for interaction with the complex. Nevertheless, themajority of the documents do not provide industrial processes butinstead the study of the improvement in the solubility of an activesubstance/cyclodextrin complex via the agent for interacting with thecomplex. They relate specifically to a listing of agents for interactionwith the complex which are tested for the same active principle, and theanalytical results obtained. Furthermore, few documents exist whichprovide complexing in a supercritical medium.

The documents describing processes for complexing with a cyclodextrin bya supercritical fluid are as follows: With the aim of fixing volatilemolecules by inclusion, Kamihira M. et al. (J. of Fermentation andBioengineering, Vol. 69, No. 6, 350-353, 1990) describe a process forthe extraction of volatile aromatic compounds and for trapping byinclusion in cyclodextrins. Geraniol and mustard oil are thus extractedby a pressurized fluid and then vaporized in dynamic mode in a secondreactor comprising cyclodextrins. The influence of the variousparameters is studied by measuring the degree of inclusion of thearomatic compounds in the cyclodextrins. However, the inclusion stage iscarried out in dynamic mode and not in static mode. Furthermore, theapplication claimed by the authors is completely different since itinvolves fixing volatile molecules by inclusion. Finally, this processis not carried out with supercritical fluids but with pressurized gases.

Van Hees et al. (Pharmaceutical Research, Vol. 16, No. 12, 1999)describe, in their publication, a process for the inclusion ofcommercial piroxicam in β-cyclo-dextrins with supercritical CO₂. Aspiroxicam is a nonsteroidal anti-inflammatory of low solubility inwater, its inclusion in β-cyclodextrins should make it possible toincrease its solubility in water. The process consists in placing amixture of piroxicam and of β-cyclodextrins. (molar ratio 1/2.5) in apressurized autoclave left in static mode. After depressurizing, themixture obtained is milled and homogenized. The complex is subsequentlydried before characterization by:

-   DSC (differential scanning calorimetry)-   Differential solubility technique-   Spectroscopic methods.

These analyses make it possible to decide on the degree of complexing ofthe piroxicam with the β-cyclodextrin. The importance of an agent forinteraction with the complex with regard to the dissolution of thecomplex thus obtained is not mentioned. Furthermore, no diffusion agentis used in the stage of formation of the complex with supercritical CO₂in static mode.

Patent Application WO 03/043604 discloses a process for the preparationof molecular complexes of active substances in host molecules. Theprocess employs a stage of molecular diffusion by bringing a densepressurized fluid into contact in static mode, in the optional presenceof a diffusion agent: water.

However, this stage is followed by an obligatory stage of washing withsupercritical CO₂. Furthermore, no agent for interaction with thecomplex is used.

Various documents form part of the improvement in the solubility of anactive substance by addition of an agent for interaction with thecomplex (Redenti E. et al, J. of Pharmaceutical Sciences, Vol. 89, 1-8,2000). The solubility of the active substance alone, or the activesubstance in the presence of the agent for interaction with the complex,of the active substance/cyclodextrin binary complex and finally of theactive substance/cyclodextrin/agent for interaction with the complexternary complex are studied. However, none of the processes describeduses supercritical CO₂ or in particular a stage of molecular diffusionin static mode using a diffusion agent.

Thus, Buvári-Barcza et al. (J. of Inclusion Phenomena and MacrocyclicChemistry, Vol. 42, 209-212, 2002) study the solubility of benzoicacid/β-cyclodextrin and benzene/β-cyclodextrin complexes in the presenceof acetic acid. The solubility of the benzene/ β-cyclodextrin complex isindependent of the concentration of acetic acid whereas that of thebenzoic acid/β-cyclodextrin complex increases with the concentration ofacetic acid. The interpretation of the authors is as follows: in themolecule/p-cyclodextrin/acetic acid ternary complex, the potentialhydrogen bonds between the molecule and the interior cavity of thecyclodextrin promote other interactions external to the cyclodextrin.

Likewise, Mura et al. (J. of Inclusion Phenomena and MacrocyclicChemistry, Vol. 39, 131-138, 2001) measure the solubility of econazolein the presence of cyclodextrins (α-, β-, γ-,hydroxypropyl-β-cyclodextrins) and of hydroxy acids (tartaric, citric,gluconic, malic and lactic acids). The ternary complexes are prepared byphysical mixing or milling of the 3 compounds, coevaporation orlyophilization of a solution comprising the 3 compounds. The formationof a ternary complex is monitored by DSC. Only lyophilization makes itpossible to obtain a DSC profile no longer exhibiting the peak for themelting of econazole.

The authors conclude that a synergistic effect is observed in theternary complex, since the solubilities observed are up to 20 timesgreater than that of an econazole/cyclodextrin binary complex.

The same authors (Int. J. of Pharmaceutics, Vol. 260, 293-302, 2003)have also studied ternary complexes ofnaproxen/hydroxypropyl-β-cyclodextrin/amino acid type. The complexesmentioned are prepared either by co-milling or by co-evaporation of awater-ethanol solution comprising the 3 compounds.

Piel et al. (J. of Pharmaceutical Sciences, Vol. 86-4, 475-480, 1997)present a study of solubility of a nimesulide/L-lysine/β- orγ-cyclodextrin complex obtained by spray drying or evaporation. Thesolubility of the ternary complex is, depending on the pH of thesolution, up to 3600 times greater than that of nimesulide alone. Hereagain, the authors speak of a synergistic effect of the cyclodextrin andof the L-lysine.

Fenivesy et al. (Proceedings of the 7th International CyclodextrinsSymposium, 414-418, 1994) are concerned with the complexing of theactive substances terfenadine, domperidone and astemizole withhydroxypropyl-β-cyclodextrin in the presence of hydroxy acids.

Two patents (EP 0 991 407 and EP 1 018 340) disclose the preparation ofactive substances/agent for interaction with the complex/cyclodextrinternary complexes. The processes employed are kneading, spray drying,evaporation or lyophilization. The process consists either in preparingthe complex of the salt of the active substance or in bringing the 3compounds into contact simultaneously during the process.

A patent (EP 0 153 998 A2), filed by Chiesi et al., discloses thepreparation of complexes of piroxicam and of β-cyclodextrin in thepresence in particular of an ammoniacal solution. However, the processused is not carried out with supercritical CO₂.

The only two documents relating to the preparation of a complex withsupercritical CO₂ in the presence of an agent for interaction with thecomplex are as follows: By following the same method as that describedabove (Pharmaceutical Research, Vol. 16, No. 12, 1999), Van Hees et al.(Journal of Inclusion Phenomena and Macrocyclic Chemistry, No. 44, pp271-274, 2002) describe the use of an agent for interaction with thecomplex, L-lysine or trometamol, in the preparation of apiroxicam/β-cyclodextrin complex with supercritical CO₂.

The use of L-lysine or of trometamol allows them both to increase thedegree of inclusion of the piroxicam in the β-cyclodextrin and toimprove the dissolution of the complex formed.

Characterization is carried out by:

-   DSC (differential scanning calorimetry)-   Differential solubility technique-   Kinetics of dissolution in a buffered medium

However, no diffusion agent is used during this stage. V. Barillaro etal. (Proceeding of the 6th International Symposium on SupercriticalFluids, Versailles, pp 1897-1902, 2003) focused more on the improvementwhich might be contributed by the addition of an acidic agent forinteraction with the complex in increasing the degree of inclusion ofmiconazole in cyclodextrins. Various agents for interaction with thecomplex (malic acid, maleic acid, fumaric acid, citric acid) and variouscyclodextrins (β-cyclodextrin, HP-β-cyclodextrin, γ-cyclodextrin,HP-γ-cyclodextrin) were used.

However, the inclusion stage is carried out in dynamic mode and not instatic mode.

In the two documents mentioned above, it is thus important to know thatthe complexing with supercritical fluids is carried out on the activesubstance/agent for interaction with the complex/cyclodextrin ternarymixture and that, furthermore, the agent for interacting with thecomplex is not arginine.

The piroxicam molecule, represented below, has an enol functional groupand a pyridine ring which may or may not be salified according to the pHvalue of the dissolution medium.

Wiseman et al. (Arzneim.-Forsch./Drug Res., 26 (7), 1976, 1300-1303)have determined, in a 2:1 (v/v) dioxane:water mixture, the pKa value ofthe enol functional group (pKa˜2) and of the pyridine ring (pKa˜6.3).

According to the pH value of the dissolution medium, the piroxicam (PX)molecule will thus exist in various forms, namely:

pH <2

-   the pyridine ring is protonated to give a pyridinium ion (NH⁺)-   the enol functional group is not ionized (—OH)

2<pH<7

-   the pyridine ring is protonated (NH⁺)-   the enol functional group is ionized to give enolate (O⁻).

This is the zwitterionic structure of piroxicam. In terms of molecularconformation, the zwitterionic structure is planar. This flatnessresults from intramolecular hydrogen bonds between the enolate anion andthe amide group, on the one hand, and then the carbonyl functional groupand the pyridinium cation, on the other hand.

pH>7

-   the enol functional group is ionized (O⁻)-   the pyridine ring is not protonated (N).

The inventors have discovered, surprisingly, that the separation of thestages of complexing and of addition of the agent for interaction withthe complex makes it possible to substantially improve thephysicochemical properties of the complex thus obtained. Furthermore,they have also noticed that the use of arginine as agent for interactionwith the complex when the active substance is piroxicam makes itpossible to obtain complexes having the most advantageous properties.

The object is to improve the in vivo dissolution of a water-insolubleactive substance, this being achieved by including the active substancein a soluble porous support and by then modifying the physicochemicalproperties of the system thus formed.

The present invention thus relates to a process for the preparation of asoluble inclusion compound comprising one or more active substancesincluded in one or more host molecules, the active substance orsubstances not being very soluble in an aqueous medium, characterized inthat it comprises the following successive stages:

-   a. bringing one or more active substances into contact with one or    more host molecules,-   b. carrying out a stage of molecular diffusion by bringing a dense    pressurized fluid into contact, in static mode, with the mixture    obtained in stage (a) in the presence of one or more diffusion    agents,-   c. recovering the active substance/host molecule molecular complex    thus formed,-   d. carrying -out a stage which consists in adding to and mixing with    the active substance/host molecule molecular complex an agent for    interaction with the complex,-   e. recovering the soluble inclusion compound thus formed.

In the process according to the present invention, there is no stage ofwashing with supercritical CO₂ between stages (c) and (d).

In another advantageous embodiment, stage (e) is followed by a stage (f)of drying the soluble interaction compound, advantageously between 60°C. and 80° C. and advantageously overnight.

The process according to the present invention is thus composed of thelinking of two phases, which are:

-   the formation of an inclusion complex between an active substance    and a host molecule in a supercritical medium (stages (a), (b) and    (c))-   the noncovalent “fixing” of an agent for interaction with the    complex to the complex obtained, in order in particular to improve    its physicochemical properties (stages (d) and (e)).

The invention thus discloses a five-stage process.

-   the first three “maturing” stages consist essentially of a phase of    molecular diffusion in a dense pressurized medium, and in particular    supercritical medium, which makes it possible to include active    substances in host molecules, in particular cyclodextrins. The    object desired during this diffusion phase is to form inclusion    complexes between active substances and the host molecule.

The term “inclusion complex” or “molecular complex” is understood tomean, within the meaning of the present invention, any complex combiningin a noncovalent fashion the active substance and the host molecule.Advantageously, it is the complex resulting from stage (b) of theprocess according to the present invention.

The term “soluble inclusion compound” is understood to mean, within themeaning of the present invention, any entity formed by the combinationof the agent for interaction with the complex, on the one hand, and themolecular complex, on the other hand. Advantageously, it is the finalproduct obtained by the process according to the present invention.

The term “agent for interaction with the complex” is understood to mean,within the meaning of the present invention, any organic or inorganicagent which improves the physicochemical properties, in particular theproperties of dissolution in an aqueous medium, of the molecular complexby interactions without covalent bonds with the active substanceincluded in the host molecule or directly with the molecular complex.Advantageously, the agent for interaction with the complex is asurfactant, for example sodium lauryl sulfate or Tween, an acid or abase. Advantageously, it is an acid or a base. The choice of an acidicor basic agent will depend on the acidic or basic nature of the activesubstance. Thus, preferably, an acidic agent will be used with amolecular complex comprising a basic active substance and a basic agentwith a molecular complex comprising an acidic active substance.

Advantageously, the agent for interaction with the complex is chosenfrom an amino acid, a carboxylic acid, an acetate, a carboxylate, anamine or aqueous ammonia, advantageously in the form of a 28% ammoniacalsolution. More advantageously still, it is chosen from acetic acid,tartaric acid, citric acid, gluconic acid, malic acid, lactic acid,maleic acid, fumaric acid, L-lysine, L-valine, L-isoleucine, L-arginineor aqueous ammonia. More advantageously still, it is aqueous ammonia.

The term “dense pressurized fluid” is understood to mean, within themeaning of the present invention, any fluid used at a temperature or apressure greater than their critical value. Advantageously, it is pureCO₂ or CO₂ in a mixture with an organic solvent conventionally used by aperson skilled in the art.

The term “active substance which is not very soluble in an aqueousmedium” is understood to mean, within the meaning of the presentinvention, any active substance which is insoluble or not very solublein an aqueous medium and which has in particular a solubility of lessthan at least 20 μg/ml. In particular, it can be a pharmaceutical activeprinciple (mention may be made, by way of examples, of analgesics,antipyretics, aspirin and its derivatives, antibiotics,anti-inflammatories, antiulceratives, antihypertensives, neuroleptics,antidepressants, oligonucleotides exhibiting a therapeutic activity,peptides exhibiting a therapeutic activity and proteins exhibiting atherapeutic activity), a cosmetic active principle or a nutraceuticactive principle. Advantageously, it is an active substance chosen fromthe group consisting of anilide derivatives, epipodophyllotoxinderivatives, minoxidil, piroxicam, valeric acid, octanoic acid, lauric-acid, stearic acid, tiaprofenic acid, omeprazole, econazole, miconazole,ketoconazole, astemizole, cyclobenzaprine, nimesulide, ibuprofen,terfenadine, domperidone, naproxen and eflucimibe; advantageously, it ispiroxicam.

The term “host molecule” is understood to mean, within the meaning ofthe present invention, any substance capable of capturing activesubstances. Advantageously, the host molecule is chosen from the groupconsisting of polysaccharides and saccharides, in particularcyclodextrins and their mixture. Advantageously, it is β-cyclodextrin,methyl-β-cyclodextrin, γ-cyclodextrin or hydroxypropyl-β-cyclodextrin.Advantageously, it is β-cyclodextrin.

Cyclodextrins are “cage” molecules as they comprise, within theirstructure, a relatively rigid and hydrophobic cavity which allows themto confine or encapsulate other molecules. The complexing phenomenon isthe consequence of a multitude of interactions (substrate/solvent,solvent/solvent and cyclodextrin/ solvent) which result in the morestable thermodynamic state:

-   (1) Van der Waals' interactions;-   (2) hydrophobic interactions;-   (3) hydrogen bonds;-   (4) the release of water molecules with a high energy during the    substitution by the guest molecule;-   (5) the release of the strain energy within the cyclodextrin    molecule during the formation of the complex.

Advantageously, the soluble inclusion compound consists of thecombination of piroxicam, of a cyclodextrin and of arginine,advantageously L-arginine.

The term “diffusion agent” is understood to mean, within the meaning ofthe present invention, any solvent which promotes an interaction of theactive substance with the host molecule.

Advantageously, this diffusion agent is chosen from the group consistingof alcohols, ketones, ethers, esters and water, with or withoutsurfactant, and their mixtures. More advantageously still, it is water.

The term “static mode” is understood to mean, within the meaning of thepresent invention, a reaction or a process in which all the reactantsare brought together simultaneously and where the reaction is allowed totake place. For example, in stage (b) of the present invention, theactive substance(s), water and supercritical CO₂ are placed in anautoclave and reaction is allowed to take place for several hours. Theweight of product does not change during reaction. Conversely, indynamic mode, the reactants are introduced as the reaction ormanufacture progresses. Often, in the case of a dynamic mode,circulation of a fluid or stirring is involved. The weight of productchanges during the manufacture.

During stage (a), the active substance and the host molecule areintroduced in solid or liquid form into a receptacle into which areinjected, during stage (b), the dense pressurized fluid and thediffusion agent in carefully chosen proportions. The pressure andtemperature conditions and the duration of the treatment are defined byany appropriate method according to the nature of the active substanceor substances and of the host molecule or molecules.

Advantageously, stage (b) of molecular diffusion of the processaccording to the present invention is carried out with stirring.

The diffusion agent can be added continuously or portionwise in anamount of between 1 and 50% by weight, preferably between 10 and 25% byweight.

The active substance/host molecule/agent for interaction with thecomplex molar ratio can be chosen so as to ensure the best inclusion ofthe active substance- in the host molecule. Thus., advantageously, theactive substance/host molecule molar ratio is between 1/1 and 1/10,advantageously between 1/1 and 1/5, advantageously between 1/2 and1/2.5, more advantageously still 1/2.5. Likewise, the activesubstance/agent for interaction with the complex molar ratio isadvantageously between 1/1 and 1/3, advantageously 1/1, moreadvantageously 1/1.2.

The time necessary for the molecular diffusion of stage (b) isdetermined by any appropriate method. This step (b) can be repeated asoften as desired in order to obtain a satisfactory rate of dissolution.

Advantageously, stage (b) lasts between approximately 1 and 16 hours.

The pressure and temperature conditions of stage (b) are chosen so as topromote molecular diffusion. Advantageously, the pressure of thesupercritical fluid is between 0.5 MPa and 50 MPa and the temperaturebetween 0 and 200° C.

Advantageously, stage (b) of the process according to the presentinvention is carried out in a closed reactor, in particular anautoclave.

The process can be carried out batchwise or continuously.Advantageously, the process according to the present invention iscarried out batchwise.

Advantageously, stage (b) of the process according to the presentinvention is carried out in a closed, optionally stirred, reactor fedwith the dense fluid and the solution of active substance, ifappropriate, continuously.

-   The final two stages ((d) and (e)) consist in adding to and mixing    with the active substance/host molecule complex an agent for    interaction with the complex. This agent for interaction with the    complex interacts according to two plausible hypotheses: strong    interactions with the active substance included in the host molecule    during the preceding stages and/or strong interactions with the    complex formed previously.

This makes it possible to improve mainly the properties of dissolutionof the complex in biological liquids and in particular water and/oroptionally to increase the degree of inclusion of the active substancein the host molecule.

The improvement in the physicochemical properties, in particular interms of dissolution, of the system formed can result from

-   a noncovalent interaction of the agent for interaction with the    complex with the active substance, the host molecule or both    (complexing, salification, and the like)-   a local variation in the pH of the dissolution medium-   a production of a system exhibiting a eutectic-   a modification of the interface between the system and its    dissolution medium (surfactant effect, particle size change).

Advantageously, stage (d) of the process is carried out in a semisolidmedium, the complex not being dissolved in an aqueous medium before theaddition of the agent for interaction with the complex. This agent willthus simply moisten the complex or form a paste with the complex.Advantageously, stage (d) is carried out with stirring.

The present invention also relates to a soluble inclusion compoundcomprising one or more active substances included in one or more hostmolecules, the active substance or substances not being very soluble inan aqueous medium, and an agent for interaction with the complexobtainable by the process according to the present invention.

Advantageously, the degree of inclusion of the active substance in thesoluble inclusion compound according to the present invention is greaterthan 95%, calculated by DSC analysis as described below, advantageouslygreater than 98%, advantageously approximately 99%.

Advantageously, the solubility of the active principle when it is foundin the form of the complex according to the present invention is greaterthan 2.5 g/l, advantageously at least equal to 3 g/l. Advantageously,the degree of dissolution of the active substance present in a 4 g/lsolution in water, measured at 38° C. after stirring for between 5 and120 minutes, is greater than 63%, advantageously at least equal to 75%.Advantageously, the active substance is piroxicam and the solubility andthe degree of dissolution are measured at pH=6.3.

Advantageously, the host molecule is cyclodextrin and the activesubstance is piroxicam.

Advantageously, piroxicam is present in the complex according to thepresent invention with a zwitterionic structure.

In the context of the present invention, the term. “degree ofdissolution” is understood to mean the percentage of piroxicam dissolvedafter stirring a mixture of water and of piroxicam at 37° C. for 5 to120 minutes. A 4 g/l mixture of piroxicam in water will generally beused to measure this degree. This dissolution can be measured by adissolution test as indicated below (dissolution test for piroxicam).

The present invention additionally relates to a complex comprisingpiroxicam, a cyclodextrin and arginine, characterized in that the degreeof dissolution of the piroxicam present in a 4 g/l solution in water,measured at 37° C. after stirring for between 5 and 120 minutes, isgreater than 90%, advantageously greater than 95%, advantageously equalto 99%.

The term “cyclodextrins” is understood to mean, within the meaning ofthe present invention, cyclodextrins, modified cyclodextrins and theirmixtures. Advantageously, the cyclodextrin is β-cyclodextrin,methyl-β-cyclodextrin, γ-cyclodextrin or hydroxypropyl-β-cyclodextrin.Advantageously, it is β-cyclodextrin.

Advantageously, the piroxicam/cyclodextrin/arginine complex according tothe present invention exhibits good crystallinity. Advantageously, thiscomplex-exhibits less than 40% by weight of amorphous phase, moreadvantageously still less than 30% by weight of amorphous phase,advantageously an amount of amorphous phase of less than or equal to 20%by weight.

Advantageously, the degree of inclusion of piroxicam in thepiroxicam/cyclodextrin/arginine complex according to the presentinvention, measured by differential scanning calorimetry as describedbelow, is greater than 98%, advantageously greater than 99%,advantageously approximately 100%.

Advantageously, piroxicam is present in the complex according to thepresent invention with a zwitterionic structure.

The piroxicam/cyclodextrin/arginine complexes according to the presentinvention are obtainable by the process according to the presentinvention as described above. However, they are also obtainable by theprocess below.

Thus, a process for the preparation of a piroxicam/cyclodextrin/argininecomplex according to the present invention comprises the followingsuccessive stages:

-   (1) bringing piroxicam into contact with a cyclodextrin and    arginine,-   (2) carrying out a stage of molecular diffusion by bringing a dense    pressurized fluid into contact, in static mode, with the mixture    obtained in stage (1) in the presence of one or more diffusion    agents,-   (3) recovering the piroxicam/cyclodextrin/arginine complex thus    formed.

This process makes it possible, in the case ofpiroxicam/cyclodextrin/arginine complexes, to obtain complexes havingspecific properties.

-   Stage (2) of molecular diffusion in static mode, referred to as    maturing stage, consists essentially of a phase of molecular    diffusion in a dense pressurized medium, and in particular a    supercritical medium, which makes it possible to include piroxicam    in the cyclodextrins. The object desired during this diffusion phase    is to form inclusion complexes between piroxicam, the cyclodextrin    and arginine.

The complex thus formed combines piroxicam, the cyclodextrin andarginine in a noncovalent manner. Arginine, which acts as interactionagent, interacts according to two plausible hypotheses: stronginteractions with piroxicam included in the cyclodextrin and/or stronginteractions with the complex formed.

The presence of arginine makes it possible to improve mainly theproperties of dissolution of the complex in. biological liquids and inparticular water. The improvement in the physicochemical properties, inparticular in terms of dissolution, of the system formed can result from

-   a noncovalent interaction of arginine with piroxicam, the    cyclodextrin or both (complexing, salification, and the like)-   a local variation in the pH of the dissolution medium-   a production of a system exhibiting a eutectic-   a modification of the interface between the system and its    dissolution medium (surfactant effect, particle size change).

The term “dense pressurized fluid” is understood to mean, within themeaning of the present invention, any fluid used at a temperature or apressure greater than their critical value. Advantageously, it is pureCO₂ or CO₂ as a mixture with an organic solvent conventionally used by aperson skilled in the art.

The term “diffusion agent” is understood to mean, within the meaning ofthe present invention, any solvent which promotes an interaction ofpiroxicam and the cyclodextrins.

Advantageously, this diffusion agent is chosen from the group consistingof alcohols, ketones, ethers, esters and water, with or withoutsurfactant, and their mixtures. More advantageously still, it is water.

The term “static mode” is understood to mean, within the meaning of thepresent invention, a reaction or a process in which all the reactantsare brought together simultaneously and where the reaction is allowed totake place. For example, in stage (2) of the present invention,piroxicam, the water, arginine and supercritical CO₂ are placed in anautoclave and reaction is allowed to take place for several hours. Theweight of product does not change during reaction. Conversely, indynamic mode, the reactants are introduced as the reaction ormanufacture progresses. Often, in the case of a dynamic mode,circulation of a fluid is involved. The weight of product changes duringthe manufacture.

Advantageously, stage (2) of molecular diffusion of the processaccording to the present invention is carried out with stirring.

In a specific embodiment of the invention, during stage (1), piroxicam,arginine and the cyclodextrins are introduced in the solid or liquidform into a receptacle into which are injected the dense pressurizedfluid and the diffusion agent in carefully chosen proportions. Thepressure and temperature conditions and the duration of the treatmentare defined by any appropriate method.

The piroxicam/cyclodextrin/arginine molar ratio can be chosen so as toprovide the best inclusion of piroxicam in the cyclodextrins. Thus,advantageously, the piroxicam/cyclodextrin molar ratio is between 1/1and 1/10, advantageously between 1/1 and 1/5, advantageously between 1/2and 1/2.5, more advantageously still 1/2.5. Likewise, thepiroxicam/arginine molar- ratio is advantageously between 1/1 and 1/3,advantageously 1/1, more advantageously 1/1.2.

The diffusion agent can be added continuously or portionwise in anamount of between 1 and 50% by weight with respect to the total weightof the mixture, advantageously between 20 and 25% by weight with respectto the total weight of the mixture.

The time necessary for the molecular diffusion of stage (2) isdetermined by any appropriate method. This stage (2) can be repeated asoften as desired in order to obtain a satisfactory rate of dissolution.

Advantageously, stage (2) lasts between approximately 2 and 16 hours,advantageously 1 hour.

The pressure and temperature conditions of stage (2) are chosen so as topromote molecular diffusion. Advantageously, the pressure of thesupercritical fluid is between 5 MPa and 40 MPa, advantageously. 15 MPa,and the temperature between 0 and 120° C., advantageously 100° C.

Advantageously, stage (2) of the process according to the presentinvention is carried out in a closed reactor, in particular anautoclave.

The process can be carried out batchwise or continuously.Advantageously, the process according to the present invention iscarried out batchwise.

Advantageously, stage (2) of the process is carried out in a closed,optionally stirred, reactor fed with the dense fluid and piroxicam, and,if appropriate, continuously.

Carrying out the stage of molecular diffusion in a dense pressurizedmedium in the presence of a diffusion agent makes possible stronginteraction of the piroxicam particles with the cyclodextrins, whichpromotes the dissolution in an aqueous medium.

The present invention additionally relates to a pharmaceuticalcomposition comprising a piroxicam/cyclodextrin/arginine complexaccording to the present invention and optionally a pharmaceuticallyacceptable excipient.

The present invention also relates to a piroxicam/cyclodextrin/argininecomplex according to the present invention or a pharmaceuticalcomposition according to the present invention as medicamentadvantageously having an anti-inflammatory action and advantageouslyintended to treat inflammatory rheumatism, polyarthritis, arthrosis,tendinitis or post-traumatic conditions of the locomotor apparatus.

The following examples are given by way of indication and withoutimplied limitation.

The various examples provided were carried out with piroxicam as activesubstance, β-cyclodextrin as host molecule and water as diffusion agent.The aqueous ammonium or arginine was used as agent for interaction withthe complex.

DESCRIPTION OF THE FIGURES

FIG. 1 represents the NMR spectrum of the complex obtained according toexample 7.

FIG. 2 represents the ROESY-intramolecular NOE spectrum of the complexobtained according to example 7.

FIG. 3 represents the TG-DTG profile (thermogravi-metric analysis) ofβ-cyclodextrin using the following process: increase in temperature of5° C. per minute up to 450° C. and then decrease in the temperature of10° C. per minute down to 80° C.

FIG. 4 represents the DSC profile of β-cyclodextrin using the followingmethod: equilibration at 0° C. followed by an increase in thetemperature of 5° C. per minute up to 240° C.

FIG. 5 represents the profile of the thermogravi-metric analysis ofL-arginine using the same process as that of FIG. 3.

FIG. 6 represents the DSC profile of L-arginine using the same processas that of FIG. 4.

FIG. 7 represents the DSC profile of piroxicam using the same process asthat of FIG. 6.

FIG. 8 represents the DSC profile of a sample of piroxicam melted at260° C. and then cooled to 0° C., using the process of equilibration at205° C., of decrease in the temperature of 20° C. per minute down to 0°C. and of increase in the temperature of 5° C. per minute- up to 220° C.

FIG. 9 represents the DSC profile of the complex obtained according toexample 7, using the process of equilibration at 0° C., of increase inthe temperature of 5° C. per minute up to 240° C. and then of decreasein the temperature of 5° C per minute down to 80° C.

FIG. 10 represents the TG-DTG profile of the complex obtained accordingto example 7, using the process of increase in the temperature of 5° C.per minute up to 450° C. and then of decrease in the temperature of 10°C. per minute down to 80° C.

FIGS. 11 to 16 represent the molecular modeling of the minimizedstructure of the piroxicam/β-cyclodextrin 1:2 inclusion complex. Inparticular, FIG. 11 represents the side view with the hydrogen atoms,FIG. 12 represents the side view without the hydrogen atoms, FIG. 13represents the view of the cavity on the benzothiazine ring side withthe hydrogen atoms, FIG. 14 represents the view of the cavity on thebenzothiazine ring side without the hydrogen atoms, FIG. 15 representsthe view of the cavity on the pyridine ring side with the hydrogen atomsand FIG. 16 represents the view of the cavity on the pyridine ring sidewithout the hydrogen atoms.

FIG. 17 represents the X-ray diffraction diagram of the complex obtainedaccording to example 7 and of the complex obtained with the process ofpatent EP 0 153 998.

ANALYSIS

The degree of inclusion of the active substance in the host molecule isevaluated by differential scanning calorimetry (DSC).

The DSC analyses of the complexes obtained according to examples 1 to 6were carried out in the following way: A temperature gradient is appliedto the product to be tested under a nitrogen stream using a Perkin-ElmerDSC 7 device.

The complexing efficiency is evaluated by measuring the reduction in (ordisappearance of) the thermal peak relative to the melting of the activeprinciple which has “remained free” in the crystalline form.

The DSC analysis of the complex obtained according to example 7 wascarried out in the following way:

The equipment, DSC Q100 TA Instruments, was calibrated using the indiummelting signal under a stream of nitrogen of 50 ml.min⁻¹. The sample isanalyzed in a hermetically sealed boat of 5° C.min⁻¹.

The thermogravimetric analyses are carried out using the TA InstrumentsTGA2950 HR equipment in order to determine the thermal stability rangeof the samples. The device is calibrated at ambient temperature andusing the Curie point of nickel at 360.46° C. The accuracy of thebalance is confirmed by the analysis of a calcium oxalate sample.

The analysis is carried out under a stream of nitrogen of 60 ml.min⁻¹from 25° C. to 450° C.

The amount of water in the samples is determined by coulometry using theMettler KF DL37 coulometer. The equipment is calibrated with the sodiumtartrate dihydrate standard (%H₂O=15.66±0.05%)

Powder X-ray diffraction is carried out with the following equipment andconditions:

-   Philips Xpert MPD diffractometer, Philips generator, voltage 40 kV,    current strength 20 mA.-   copper anticathode (K_(a)=1.5418367 angstrom), Ni filter-   ⅛ entry slit-   Xcelerator detector-   continuous scan mode-   sample ground to a powder on a plate-   angular range (°2θ) 4 to 100-   scan time: 80 seconds

Phase analysis is carried out using Visual CRYSTAL software.

The Raman scattering spectra are obtained with the following equipmentand conditions:

-   Jobin Yvon LabRAM HR 800 Raman spectrometer-   Temperature: 22° C.-   Samples: powder on microscope coverglass-   Exciting wavelength: 752 nm, laser power 10 mW on the sample-   Spectral resolution 2 cm⁻¹, scattering volume approximately 1 μm³    (grating 600 lines)

The proton NMR spectrum is recorded on a Bruker Avance DPX spectrometerat the nominal frequency of 400 MHz using a broad band inverse probeequipped with a field gradient accessory along the Z axis. Thespectrometer is locked beforehand on the resonance frequency of thedeuterium of the dissolution solvent, in this case d₆-dimethylsulphoxide (Eurisotop, ref. D 310B, batch A 2731). The chemical shiftsare given in ppm with respect to TMS (tetramethylsilane), used asinternal standard.

The ROESY (Rotating frame Overhauser Enhancement SpectroscopY) spectrumis obtained by applying the Bruker pulse microprogram “roesytp.2”.

Recording is carried out in phase-sensitive mode with 1024 incrementsand 72 scans per increment, i.e. a total experimental time of 53 hoursper product. Acquisition is carried out by selecting a spectral windowof 6410.256 Hz, a relaxation time of 2 s and a spin lock time of 350 ms.Prior to the recording of the spectrum, the sample is correctly degassedin order to observe the maximum intra- and intermolecular NOE effect.

Due to the low solubility of the complexes in water and to thesensitivity of the NMR spectrometer in our possession, we preferred towork in deuterated dimethyl sulphoxide, in which it is possible toexpect a concentration of complex of 2% (w/v).

Molecular modeling: the optimizing of the piroxicam/β-cyclodextrin (1:2)inclusion complex is carried out with Hyperchem®, version 6.02, software(Hypercube, Gainsville, USA), implemented on an HP Vectra model PentiumIII personal computer.

The molecular structure of the piroxicam used for the minimizationcalculations is taken from the publication by Jon Bordner et al. (ActaCryst., 1984, C40, 989-990).

The molecular structure of the β-cyclodextrin originates from theCambridge Crystallographic Database (M. R. Caira et al., J. Chem. Soc.,Chem. Commun., 1994, 1061-1062).

The energy minimization of the geometry and of the conformation of thecomplex is carried out using the MM2 force field.

The simulation of the physicochemical propeties (pKa, logD andsolubility) from the piroxicam molecule is carried out with the ACD/LogDSuite software (ACD/Labs software, Toronto, Canada).

In order to measure the dissolution properties of the powder, theequivalent of 4 g/l of piroxicam is dissolved in an aqueous solution at37° C. After 15 min, a sample is taken and then the amount of piroxicamdissolved is measured by HPLC. The result is expressed in grams ofpiroxicam dissolved per liter of water. According to this method, thesolubility of pure piroxicam in water is less than 0.2 g/l.

Piroxicam Dissolution Test

Procedure:

The assaying of piroxicam in the dissolution solution is carried out byHPLC:

Equipment used:

Waters HPLC system:

-   2695 separation module,-   2487 UV detector.

Chromatographic conditions:

Column: Waters X-Terra MS C18 250×4.6 mm.

1.1.1.1.1.1 Mobile phase: Route A

60% buffer pH=3.5 KH₂PO₄ 6.81 g/l adjusted to the pH with H₃PO₄ dilutedR,

40% acetonitrile.

Flow rate: 1 ml/min

Detector wavelength: 230 nm

Sensitivity of the detector: 2 AUFS

Volume injected: 20 μl

Oven temperature: 40° C.

Analysis time: 12 minutes

Preparation of the control solutions:

Control solution: SM: 50 mg of control piroxicam are

introduced into a 100 ml flask. Dissolution is carried out with 20 ml ofdimethylformamide and the solution is made up to volume with methanol.

Range:

T1: Dilution of T3 to 1/20^(th) in 40 acetonitrile/60 water. T2:Dilution of T3 to 1/10^(th) in 40 acetonitrile/60 water.

T3: Dilution of SM to 1/100^(th) in 40 acetonitrile/60 water.

T4: Dilution of SM to 1/50^(th) in 40 acetonitrile/60 water.

T5: Dilution of SM to 1/20^(th) in 40 acetonitrile/60 water.

Operating conditions for the kinetics of dissolution at 4 g/l:

Implemention of the test:

Operating conditions:

A test sample equivalent to 200 mg of piroxicam is

introduced into a 100 ml Erlenmeyer flask. 50 ml of water are added. Themixture is stirred magnetically at 400 revolutions per minute in a baththermostatically controlled at 37° C.±2° C. A 2 ml sample is withdrawnat 5, 30, 60 and 120 minutes while stirring magnetically. The widthdrawnsamples are filtered through a 0.45 μm Gelman GHP Acrodisc polypropylenefilter. The solution must be clear. The withdrawn sample is diluted to1/200^(th) in the mobile phase.

Methodology, expression of the results:

20 μl of each control solution are injected.

A linear regression is carried out on the areas of the piroxicam peakswith respect to the concentrations. The correlation coefficient must begreater than 0.995.

20 μl of the solutions to be examined are injected.

The area of the peak of piroxicam in each solution to be examined ismeasured.

The concentration X in μg/ml is deduced therefrom using the regressionstraight line of the controls.

The concentration of dissolved piroxicam in μg per ml is calculated bymultiplying by the inverse of the dilution carried out (i.e.: 200).

The degree of dissolution of the piroxicam is calculated by dividing theconcentration of dissolved piroxicam by the total concentration ofpiroxicam in the starting solution.

EXAMPLE 1 Results Obtained Using the Process According to the PresentInvention

8 grams of piroxicam, 76 grams of β-cyclodextrin and 25.2 g of water aremixed and introduced into a two liter reactor. Carbon dioxide issubsequently introduced into the reactor under a pressure of 200 bar andunder a temperature of 150° C. These operating conditions are maintainedfor a time of two hours.

After the “maturing” stage, a portion of the powder collected (12 g) ismixed with 2.11 g of a 28% ammoniacal- solution and then placed in aventilated oven at 60° C. overnight.

The DSC analysis reveals a degree of inclusion of the piroxicam in thecyclodextrin of 99%; the dissolution of the piroxicam is 3.019 g/l.

COMPARATIVE EXAMPLE 2 Results Obtained After the Maturing Stage withoutAddition of Agent for Interaction with the Complex

4 grams of piroxicam, 38 grams of β-cyclodextrin and 8.95 grams of waterare mixed and introduced into a two liter reactor. Carbon dioxide issubsequently introduced into the reactor under a pressure of 150 bar andunder a temperature of 150° C. These operating conditions are maintainedfor a time of two hours.

After reducing the medium in pressure, a portion of the powder collectedis placed in a ventilated oven at 60° C. overnight. The DSC analysisreveals a degree of inclusion of the piroxicam in the cyclodextrin of80%; the dissolution of the piroxicam is 0.246 g/l.

COMPARATIVE EXAMPLE 3 Results Obtained if the Agent for Interaction withthe Complex is Present During the Maturing Stage (b)

2 g of piroxicam, 19 g of β-cyclodextrin, 3.75 g of water and 1.5 g of28% ammoniacal solution are mixed and introduced into a two literreactor. Carbon dioxide is subsequently introduced into the reactorunder a pressure of 200 bar and under a temperature of 160° C. Theseoperating conditions are maintained for a time of two hours.

After reducing the medium in pressure, the powder collected is placed ina ventilated oven at 60° C. overnight. The DSC analysis reveals a degreeof inclusion of the piroxicam in the cyclodextrin of 50%; thedissolution of the piroxicam is 1.07-5 g/l.

COMPARATIVE EXAMPLE 4 Results Obtained if an Attempt is made to Attachthe Agent for Interaction to the Host Molecule before the Maturing Stage(b)

19 g of β-cyclodextrin, 2.11 g of 28% ammoniacal solution and 3.15 g ofpurified water are mixed and placed in a ventilated oven at 60° C.overnight. 2 g of piroxicam and 6.57 g of water are subsequently added.The mixture is subsequently introduced into a two liter reactor. Carbondioxide is subsequently introduced into the reactor under a pressure of200 bar and under a temperature of 160° C. These operating conditionsare maintained for a time of two hours.

The DSC analysis reveals a degree of inclusion of the piroxicam in thecyclodextrin of 92%; the dissolution of the piroxicam is 0.23 g/l.

COMPARATIVE EXAMPLE 5 Results Obtained if an Attempt is made to Attachthe Agent for Interaction to the Active Substance before the MaturingStage (b)

2 g of piroxicam and 2 g of 28% ammoniacal solution are mixed and placedin a ventilated oven at 60° C. overnight. 14.3 g of β-cyclodextrin and3.3 g of water are subsequently added. The mixture is subsequentlyintroduced into a two liter reactor. Carbon dioxide is subsequentlyintroduced into the reactor under a pressure of 150 bar and under atemperature of 150° C. These operating conditions are maintained for aperiod of two hours.

The DSC analysis reveals a degree of inclusion of the piroxicam in thecyclodextrin of 56%; the dissolution of the piroxicam is 1.370 g/l.

EXAMPLE 6 Results using the Process According to the Present Inventionon Batches of Semi-industrial Size: 12.5 kg

1.1 kg of piroxicam, 10.6 kg of β-cyclodextrin and 1.3 kg of water aremixed and introduced into a 50 liter reactor. Carbon dioxide issubsequently introduced into the reactor under a pressure of 150 bar andunder a temperature of 100° C. These operating conditions are maintainedfor a time of two hours.

A portion of the powder is placed in an oven at 80° C. overnight.

The DSC analysis reveals a degree of inclusion of the piroxicam in thecyclodextrin of 89%; the dissolution of the piroxicam is 1.937 g/l.

The powder collected (12.6 kg) is mixed with 2.6 kg of a 28% ammoniacalsolution and then placed in a ventilated oven at 60° C.

The DSC analysis reveals a degree of inclusion of the piroxicam in thecyclodextrin of 100%; the dissolution of the piroxicam is greater than 3g/l.

Summary of the Results Obtained

Table 1 below collates the results of the various examples 1 to 6indicated above and makes it possible to observe the added value of theprocess according to the present invention.

TABLE 1 Process/Materials Comp. Comp. Comp. Comp. Materials involved: /Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Ex. 6 Piroxicam X X X X X X Piroxicammixed X beforehand with the agent for interaction with the complexβ-Cyclodextrin X X X X X β-Cyclodextrin X mixed beforehand with theagent for interaction with the complex 1st phase: X X X X X Complexingin super- critical CO₂ medium 2nd phase: Addition X X of the agent forinteraction with the complex 1 single phase: X Complexing with the agentfor interaction with the complex in super- critical CO₂ medium Degree ofcomplexing (DSC) / 99% 80% 50% 92% 56% 100% Dissolution (g/l) <0.2 3.020.25 1.07 0.23 1.37 >3

EXAMPLE 7 Piroxicam/β-cyclodextrin/arginine Complex

43 grams of piroxicam, 384 grams of β-cyclodextrin and 25 grams ofarginine are introduced into a reactor, along with 61 grams of diffusionagent (water). Carbon dioxide is subsequently introduced into thereactor under a pressure of 15 MPa and under a temperature of 100° C.These operating conditions are maintained for a time of one hour.

The kinetics of dissolution and the degree of dissolution are measuredon the complexes obtained as indicated above in the “PIROXICAMdissolution test”. The results are collated in table 2 below.

Time Piroxicam concentration Degree of dissolution (minutes) (μg/ml) (%)5 3863 96.6 30 3854 96.4 60 3941 98.5 120 3962 99

NMR:

The ¹H NMR spectrum of a complex obtained according to example 7(FIG. 1) is in agreement with the chemical composition of this compound.

The signal observed at 13.5 ppm is attributed to the pyridinium proton,providing additional proof of the existence of a zwitterionic structureof the piroxicam in the complex obtained according to example 7.

Analysis of the ROESY spectrum (FIG. 2)

This study, which makes it possible to study the transfers ofmagnetization between protons close in space, will enlighten us on theform of inclusion of the piroxicam in the β-cyclodextrin and will thusallow us to model the corresponding complex.

From the ROESY chart, the intramolecular NOE effects (between protons ofthe same molecule) are distinguished from the intermolecular NOE effects(between protons belonging to different molecules).

While the intramolecular NOE effects are, of course, instructive interms of conformation analysis, we will mention only the intermolecularNOE effects, which provide evidence of an encapsulation of the piroxicamin the β-cyclodextrin.

The analysis of the ROESY spectrum, recorded on a complex obtainedaccording to example 7 (FIG. 2), clearly shows cross-relaxation peaksbetween H-9/10 of the piroxicam and H-5′ of the PCD and then betweenH-7, H-6 and H-3 of the piroxicam and H-3′ of the β-cyclo-dextrin. Theseresults are in agreement with a 1:2 piroxicam:β-cyclodextrin inclusioncomplex.

Thermal analysis and assaying of water: The thermogravimetric analysisof β-cyclodextrin (FIG. 3) demonstrates two transitions at 70.6° C. and313° C. The first corresponds to a loss in weight of 13.4%, in agreementwith the amount of water determined by coulometry (14.1%), and thesecond transition is attributed to the thermal decomposition of theβ-cyclodextrin.

The DSC analysis (FIG. 4) shows a broad endotherm centered on 162° C.corresponding to the dehydration phenomenon. The thermogravimetricanalysis of arginine (FIG. 5) shows that the latter is stable up toapproximately 230° C. before decomposing. Two endotherms are observed onthe DSC profile (FIG. 6), the first at 215.6° C. (melting or phasetransition) and the second at 234° C., related to the decompositionphenomenon. The DSC profile of piroxicam shows that melting occurs at201° C. with an enthalpy of 104 J.g⁻¹ (FIG. 7).

Heating/cooling cycles are carried out in which a piroxicam sample ismelted at 205° C. and then cooled to 0° C. The DSC profile of thissample (FIG. 8) reveals the existence of a glass transition at 62° C.,characteristic of the amorphous state, which crystallizes at 122° C. Aweak endotherm at 178° C., followed by an exotherm and then by a finalendotherm at 197° C. reflects the existence of several crystalline formsof the piroxicam (polymorphism).

The two endotherms at 178° C. and 197° C. would be attributedrespectively to the melting of the forms III and II, while the initialsample before the heat treatment would correspond to the form I (meltingat 201° C.) (F. Vre{hacek over (c)}er, S. Sr{hacek over (c)}i{hacek over(c)}i and J. {hacek over (S)}mid-Korbar, International Journal ofPharmaceutics, 68 (1991), 35-41).

The water content of the complex obtained according to example 7 is ofthe order of 10% w/w, as indicated in the following table 3:

TABLE 3 Result of the assays of water in the complex of example 7Complex H₂O content (TGA) H₂O content (coulometry) Obtained according9.99% 10.14% to example 7

The DSC profile of the complex obtained according to example 7 (FIG. 9)does not show the transitions at 198-200° C. and at 218° C., suggestingan inclusion of approximately 100%.

The TG DTG profile of the complex obtained according to example 7 (FIG.10) shows a transition at 191° C. This transition is attributed to thedecomposition of the arginine. This is because the first stage ofdecomposition of the arginine corresponds to a loss in weight of theorder of 39.2% and, if the content of arginine in the complex is takeninto account, approximately 6% w/w (determination by NMR), this loss inweight should be of the order of 2.35% w/w, in excellent agreement withthat observed on the TG and DTG profiles at 191° C. (2.40%).

Molecular modeling: The minimized structure of thepiroxicam:β-cyclodextrin (1:2) inclusion complex is represented in FIGS.11 to 16. This optimized structure takes into account the spatialinteractions observed by ROESY spectrometry.

As is indicated in the publication by G. M. Escandar (Analyst, 1999,124, 587-591), the piroxicam molecule is too bulky (approximately 6×13.7Å) to be completely encapsulated in a β-cyclodextrin cavity. With aPX:(βCD)₂ complex, the piroxicam molecule is completely included.

Simulation of the physicochemical properties: variation in solubility ofthe piroxicam as a function of the pH of the dissolution medium. Thezwitterionic structure presents between pH 2 and 6 the lowestsolubility. For its part, the logD value changes in the opposite sense.In the pH range 2-6, the molecule is relatively hydrophobic, whichrepresents another interaction favorable to the encapsulation. BetweenpH 6 and pH 7.5, the zwitterion is still present and the solubilityvalue increases substantially as a function of the pH, hence theinterest in preparing complexes in this pH region in order to combinethe two solubilizing effects, which are the encapsulation, on the onehand, and the salification of the piroxicam, on the other hand. The pHvalues measured are shown below: complex according to example 7 at 13.9mg/10 ml H₂=pH 7.87. X-ray diffractions of the complex obtainedaccording to example 7 and of the complex obtained according to theprocess disclosed in EP 0 153 998: the diffraction diagram isrepresented in FIG. 17. The powder diffraction diagram of the complexaccording to example 7 shows intense and very well resolved diffractionlines, providing evidence of better crystallinity of this sample incomparison with that of the complex according to the process of patentEP 0 153 998. According to the Visual CRYSTAL software, the complexobtained according to example 7 exhibits between 16 and 20% by weight ofamorphous phase.

EXAMPLE 8 Piroxicam/β-cyclodextrin/arginine Complex

400 grams of piroxicam, 3832 grams of β-cyclodextrin and 253 grams ofarginine are introduced into a reactor, along with 613 grams ofdiffusion agent (water). Carbon dioxide is subsequently introduced intothe reactor under a pressure of 15 MPa and under a temperature of 100°C. These operating conditions are maintained for a time of one hour.

The kinetics of dissolution and the degree of dissolution are measuredon the complexes obtained as indicated above in the “PIROXICAMdissolution test”. The results are collated in table 3 below.

Time Piroxicam concentration Degree of dissolution (minutes) (μg/ml) (%)5 3953 98.8 30 3895 97.3 60 3952 98.8 120  4041* 100 *Result consistentwith the uncertainty related to the measurement.

What is claimed is:
 1. A process for the preparation of an aqueoussoluble inclusion compound comprising one or more active substancesincluded in one or more host molecules, the active substance orsubstances not being very soluble in an aqueous medium, wherein itcomprises the following successive steps: a. bringing one or moreelectrically uncharged active substances into contact with one or morehost molecules, b. carrying out a step of molecular diffusion bybringing a dense pressurized fluid into contact, in static mode, withthe mixture obtained in step (a) in the presence of water as a diffusionagent in an amount of between 1 and 50% by weight with respect to thetotal weight, c. depressurizing and recovering the active substance/hostmolecule molecular complex thus formed, d. carrying out a step whichconsists of adding to and mixing with the active substance/host moleculemolecular complex ma agent for interaction with the complex underatmospheric pressure in a semi-solid medium wherein said agent fbrinteraction with the complex is an acid or a base, e. recovering theaqueous soluble inclusion compound thus formed, wherein the aqueoussoluble inclusion compound is in a salt form comprising said activesubstance, host molecule, and agent for interaction, and wherein saidhost molecule is selected from the group consisting of cyclodextrins andmixtures thereof and said dense pressurized fluid is carbon dioxide. 2.The process as claimed in claim 1, wherein the agent for interactionwith the complex is an amino acid, a carboxylic acid or aqueous ammonia.3. The process as claimed in claim 1, wherein the active substance is apharmaceutical active principle, a cosmetic active principle or anutraceutic active principle.
 4. The process as claimed in claim 3,wherein the active substance is chosen from the group consisting ofanilides, epipodophyllotoxins, minoxidil, piroxicam, valeric acid,octanoic acid, lauric acid, stearic acid, tiaprofenic acid, omeprazole,econazole, miconazole, ketoconazole, astemizole, cyclobenzaprine,nimesulide, ibuprofen, terfenadine, domperidone, naproxen andeflucimibe.
 5. The process as claimed in claim 1, wherein the pressureof the dense fluid is between 0.5 Mpa and 50 MPa and the temperaturebetween 0 and 200° C.
 6. The process as claimed in claim 1, wherein step(b) of molecular diffusion is carried out with stirring.
 7. The processas claimed in claim 1, wherein the water as the diffusion agent is addedcontinuously or portionwise.
 8. The process as claimed in claim 1wherein the agent for interaction is chosen from the group consisting ofacetic acid, tartaric acid, citric acid, gluconic acid, malic acid,lactic acid, maleic acid, fumaric acid, L-lysine, L-valine,L-isoleucine, L-arginine and aqueous ammonia.